Although B. anthrasis (anthrax), B. cereus (foodborne gastrointestinal disease), and B. thuringiensis (biological pesticide) produce a variety of pathological effects, the three bacilli are related genetically with some authors placing these organisms as subspecies of the group Bacillus cereus (Turnbull, P.C.B.1999. Definitive identification of Bacillus anthrasis-a review, Journal of Applied Microbiology, Vol. 87 pages 237-240; Helgason, E. et al. 2000. Bacillus anthrasis, Bacillus cereus, and Bacillus thuringiensis-one species on the basis of genetic evidence, Applied and Environmental Microbiology, Vol. 66 pages 2627-2630). An easy and rapid separation of these three bacterial strains is important for determining the causative agent of a pathological effect, especially with regards to the potential use of B. anthrasis as a biological weapon.
Traditionally, Bacillus cereus/Bacillus thuringiensis have been presumptively isolated from a variety of sources including foods and the environment using mannitol egg yolk polymyxin agar (MYP) dependent on expression of lecithinase activity, fermentation of mannitol and resistance to polymyxin (Compendium of Methods for the Microbiological Examination of Foods, 1992, Chapter 35, American Public Health Association). With the shortcomings of MYP involving frequent false positive and negative reactions and coalescing of colonies causing difficulty in colony enumeration, in 2001 a plating medium using 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate to detect phosphatidylinositol-specific phospholipase C (PI-PLC) in B. cereus/B. thuringiensis producing turquoise colonies was developed and patented (Peng, H. et al. 2001. Isolation and enumeration of Bacillus cereus from foods on a novel chromogenic plating medium, Food Microbiology, Vol. 18 pages 231-238; Restaino, L. 2001. Plating media for the presumptive identification of Bacillus cereus and Bacillus thuringiensis, U.S. Pat. No. 6,284,517). For B. anthrasis, blood agar containing polymyxin B with incubation at 37° C. for 24 hours has traditionally been used resulting in a large percentage of false positive isolates.
Although B. cereus, B. thuringiensis; and B. anthrasis produce PI-PLC, the molecular weight of this enzyme is different in B. anthrasis compared with the enzyme produced by the other two bacilli indicating a different mechanism of action (Guttmann, D. M. and D. J. Ellar. 2000. Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors, FEMS Microbiology Letters, Vol. 188 pages 7-13). This reaction can be demonstrated on plating medium containing the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate where after incubation B. cereus and B. thuringiensis produce turquoise colonies and B. anthrasis yield white colonies. However, phosphatidylcholine-specific phospholipase C (PC-PLC) enzyme is identical in B. cereus, B. thuringiensis and B. anthrasis, but the rate of production is slower for B. anthrasis (Guttmann, D. M. and D. J. Ellar. 2000. Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors, FEMS Microbiology Letters, Vol. 188 pages 7-13).